The long range goal of this proposal is to understand the role that tight junctions play in regulating the outer blood-retinal barrier. Malfunction of the blood-retinal barrier is implicated in many retinopathies. This proposal examines how immature, leaky, tight junctions are converted into a low permeability form, and how this process is regulated by the neural retina. During development, tight junctions of the retinal pigment epithelium (RPE) undergoes this conversion to form the outer blood-retinal barrier. Tight junctions are semi-selective, and that selectivity can be regulated. Consequently, epithelial tight junctions vary in their permeability to solutes of different size and different charge. This laboratory devised an innovative culture model that shows permeability is partially regulated by secretions of the neural retina. Those experiments led to the novel finding that junction selectivity involves multiple mechanisms. It is now imperative to define these mechanisms to understand the different regulatory pathways that affect tight junctions. This proposal addresses three hypotheses to define these mechanisms and to begin dissecting the regulatory pathways: 1) Tight junctions use size and charge discrimination to regulate diffusion across them, and these mechanisms are regulated independently by distinct pathways. This will be tested electrophysiologically and by the permeation of charged and uncharged monosaccharides. With a clear understanding of these mechanisms, it becomes possible to examine how they are regulated. 2) The phosphorylation of the tight junction proteins, occluding and 7H6, is part of regulatory mechanism that modulates specific functions of tight junctions. This will be tested by using 32/P orthophosphate to metabolically label RPE that is cultured in presence or absence of stimulatory retinal factors. These experiments will dissect the role of phosphorylation in different regulatory pathways. This will be tested with dominant negative mutants that are designed to compete with the native protein for regulatory factors. The mutations will be expressed by a retroviral expression system in primary cell culture. The regulation of the tight junction has proved to be enigmatic. Recent advances in the development of reagents coupled with this novel culture model will advance our understanding of this essential structure of the outer blood-retinal barrier.
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